1. Field of the Invention
The present invention relates to techniques for restoration of living biological tissues or promoting cell proliferation as well as to agents for growing, extending, promoting growth of hair or for restoring skin lesions and a method of promoting restoration of skin lesions.
2. Description of the Related Art
Recently, efforts have been becoming increasingly active in the field of medical techniques for regenerating a part of or particular cells of human tissues or organs for the purpose to supplement or restore the tissues or organs suffering from heavy diseases or destructive injures or lesions and regarded as difficult to be functionally restored.
Efforts and trials for regenerative medical techniques are still in the stage of animal experiments, and the techniques for regenerating human tissues have been put into practical use still only in the field of cultured skin.
In the methods now under development for regenerating tissues or organs, the technique has been employed for treating or culturing objects to be regenerated such as ES cells, lobar stem cells, or other similar tissue cells together with various types of administered stimulating agents or feeder cells.
However, the problem in the prior art is that divided cells are hardly regenerated. In other words, in the case of highly diving cells/tissues in a living body such as central nerves, heart, or kidney, once injured, the cells or the tissues are hardly regenerated by any conventional technique. On the other hand, among the various techniques for achieving the object as described above, there is the technique for separating and purifying growth factors acting in the S phase of the cell cycle for promoting DNA synthesis from a tissue of a living body. It has been known as the representative examples that the epidermal growth factor (EGF) and the hepatocyte growth factor (HGF) are effective in promotion of DNA synthesis. Other known growth factors are, for example, the insulin-like growth factor-1 (IGF-1), the insulin-like growth factor-2 (IGF-2), the transforming growth factor-a (TGF-a), and the like, and any of the factors plays an important biological role in promotion of cell proliferation.
The cell division promoting agents such as EGF and HGF conjugate to specific receptors to start cascading of protein kinase. Namely the cell division promoting agents act to the MEK (MAP kinase kinase=MAPK-ERK kinase) because of phosphorylation and activation of the MAP (mitogen-activated protein) kinase, and the MEK phosphorylate other MAP kinases, namely p44 (ERK-1: extracellular signal-regulated kinase 1) and p42 to activate the factors. Then the p42 (ERK-2) sends signals for proliferation and division to cytoplasm and cell nuclears. For instance, this MAP kinase cascade reaction is a key signaling pathway in control over a cell cycle of a live cell. It is well known that activation of ERKs by a growth factor leads to DNA synthesis when culturing a rat liver cell belonging to the first generation. Phosphatidylinositol 3-kinase (PI3K) and protein kinase B (PKB, Akt) which is a signaling kinase in the downstream region, are main control factors for survival of cells reacting to a growth factor. Recently, it was reported that activation of PKB serine—threonine kinase is involved in phosphorylation or inactivation of pro-apoptosis proteins such as BAD or caspase-9. Thus, the growth factors plate an important role in control over proliferation and survival of cells. As described above, it is generally known that a growth factor conjugates to a specific receptor of a cell to promote cascading of protein kinase initiating from the MAP kinase, and induces DNA synthesis in the cell, control over the cell cycle, regeneration of the cell, and activation of the proliferating capability of the cell such as compensatory hypertrophy.
On the other hand, ANP (atrial natriuretic peptide) is a peptide hormone presenting the strong diuretic effect by sodium secreted mainly from atrial and the angiactatic and hypotensive effects, and is classified to the three types of type α, type β, and type γ according to a difference in the molecular weight. The ANP-α is a single polypeptide chain consisting of 28 amino acid molecules and has a disulfide bond in the molecule [Cys(7)-Cys(23)] (Biochem. Biophys. Res. Commun., 118, 131-139, 1984). ANP-β is a dimmer in which two molecules of the ANP-α exist side by side but in the reverse directions. Also it has been suggested that ANP-γ is a high molecular weight protein 126 amino acid molecules contain the type a sequence in the terminal region which is a precursor in biosynthesis (Nature, 313, 397-400, 1985).
As reports of agents capable of modifying or inhibiting various physiological activities of ANP to various organs by competing with ANP for the reaction of bonding to a receptor, there are those, for instance, concerning the synthetic ligand C-ANP deleting portions of the N terminal, C terminal and a circular structure of a rat ANP (Science, 238, 675-678, 1987) or the analog III of a dimmer of human ANP [7-28] in which the ANP molecules exist side by side but in the reverse directions in which a disulfide bond is substituted with L-α-aminosuberic acid (FEBS Lett., 248, 28-34, 1989).
On the other hand, the present inventors have discovered that the ANP has the biological activities such as the effect for promoting proliferation of a chicken embryo cardiac muscular cell, but it has not been clarified whether the ANP family molecules have any effect for regenerating or repairing cells or tissue organs, over the expansive myocardial diseases, or to skin and hair.
It has generally been recognized that an action of an endocrine hormone functions and is expressed because a molecule released from a secreting organ reaches a receptor cell within the target organ and delivers a signal into the cell.
However, recently it was clarified that a unified local secretion and reaction system even in a micro tissue environment and various physiolo-pathogenic functions of a living body are adjusted by the local system.
Generally, the local renin-angiotensin-aldosterone system has been analyzed well, and it has been recognized as a therapeutic mechanism that the ACE inhibiting agent, ARB, and aldosterone inhibiting agent function not only as a hypotensive agent, but also via expression of local tissue genes in the cardiovascular system.
As for the NP (natriuretic peptide), there have been known three types of receptors, and there is the possibility that the NP functions locally. As for structures of the receptors belonging to the types A and B, it is generally recognized that the structures have an extracellular ligand-conjugating site, a site homologous to intracellular protein tyrosine kinase, and an adenylate guanylase site, and that the structures produce a cyclic GMP when the ANP family is conjugated to and cause subsequent cellular reactions. It is said that the receptor belonging to the type C has a role for clearance by fetching NP into a cell and destructing the ANP family therein, but the functions described above have not sufficiently been clarified.
We have examined formation of the cardiovascular system centering on the ANP gene by analyzing generation of growth of an embryo in the developing stage, and found out occurrence of cell reactions different from those via any known signal path due to administration of ANP. Namely, we have found cell proliferation in succession to rapid appearance of a quantity of small molecules each having a tyrosine phosphorylate residue.
Recently, it was found that the ANP genes are controlled by transcription factors such as GATA4, CSX, and TBX5, and that expression of the ANP genes explosively increases in association of growth of the cardiac system, but there are still many unknown matters relating to this phenomenon, and functions of the ANP family molecules in tissues outside the cardiac system are little known.
The NP is conventionally used as a drug for treating cardiac failures because the ANP family has the blood expansion effect via cGMP as a second messenger. However, if only the reactions via the cGMP are pharmaceutical effects of the ANP family, the efficacy must be similar to that of a nitrous agent, but the effects and actions of the ANP are clearly different from those of a nitrous agent. In addition, it is now known that distribution of ANP family receptors is not limited only the cardiovascular system, but also extends eve to the neural system, the genital system, renal, adrenal, and even to cartilago, but physiological roles of the ANP family receptors are still unknown. Furthermore, expression of the ANP genes which can be recognized in the developmental stage can be recognized only after decision of orientation of cell division, and the ANP genes are expressed frequently after cell division is started and in the phase where cell proliferation is very active. The present inventors consider that the ANP family receptors are distributed mainly in cell of mesodermal origin and are involved in proliferation or restoration of cells like autocrine or paracrine.
Based on the findings and recognitions as described above, we suppose that ANP has significant physiological functions not only the cardiovascular system, but also in cells of mesodermal origin and even in those of ectodermal origin, and are now concentrating on the study for demonstrating the assumption.